Nucleophile-reactive sulfonated compounds for the (radio)labelling of (bio)molecules; precursors and conjugates thereof

ABSTRACT

Nucleophile-reactive sulfonated compounds used as precursors to (radio)labelled (bio)molecules are produced by pre-introduction of a nucleophilic compound R* through an unusual nucleophile-induced ring-opening reaction of the sultone moiety of the precursor. The precursors and compounds conform to respective formulae (Ip) and (I): 
                         
Also disclosed are methods for producing these precursors and compounds, as well as for conjugation of these compounds with (bio)molecules, and to the drugs obtained by this method.

FIELD OF THE INVENTION

The field of the invention is that of (radio)labelling of complex andfragile (bio)active (macro)molecules, such as peptides, proteins,(bio)polymers, antibodies, oligonucleotides, nucleic acids,carbohydrates, lipids, used as (radio)pharmaceuticals.

The invention particularly relates to new labelling compounds capable ofconjugation with these bioactive (macro)molecules. These labellingcompounds form functionalised prosthetic group carrying a chosenradionuclide, in the case of radiolabelling.

The invention also concerns some new precursors of these radiolabellingcompounds, the synthesis methods of these precursors, compounds andconjugates (labelling strategies) as well as the use thereof, preferablyof the radiolabelled ones, in therapy (nuclear medicine i.a.) and/or indiagnosis (nuclear imaging i.a.) depending on the radioelement.

Diagnostic probes for medical imaging, especially by PET (PositronEmission Tomography), SPECT (Single Photon Emission Computed Tomography)or NIRF (Near-infrared fluorescence), are notably contemplated in thisinvention.

BACKGROUND ART

The radiolabelling of (bio)molecules with Fluorine-18 (¹⁸F), is widelyspread. A common PET diagnostic probe is [¹⁸F]-labeledfluorodeoxyglucose ([¹⁸F]-FDG):

-   [¹⁸F]-FDG is widely used for early detection of cancer.-   [¹⁸F]-FDG is routinely obtained by direct labelling of a mannose    derivative via nucleophilic substitution.

Similarly to the carbohydrates, the peptides, proteins and the largestpart of biomolecules contain numerous labile protons due to the presenceof different functional groups such as hydroxyls, amides, amines,thiols, acids. However, for these kinds of macromolecules, a chemicalprotection of all of the functional groups cannot always be envisaged.Therefore, most often the direct labelling of these (bio)molecules bydirect nucleophilic substitution cannot be performed.

The introduction of a radionuclide such as Fluorine-18 into a(bio)macromolecule of different nature, e. g. peptides, proteins,oligonucleotides, oligosaccharides is most often carried out via aprosthetic group bearing the radioisotope. This approach then involvesthe preparation of a functionalized and radiolabelled prostheticcompound followed by its conjugation with a specific reactive functionof the (bio)macromolecule. This strategy has the advantage of making itpossible to use severe conditions for the preparation of theradiolabelled prosthetic compound followed by its conjugation to themacromolecule under mild conditions preserving the integrity of themacromolecule.

A certain number of prosthetic compounds (also called prosthetic groups)labelled with Fluorine-18 are described in the literature. They can beclassified according to their own reactive function and/or according tothe reactive function present on the macromolecule they will react with(amines, hydrazines, oximes, acids, aldehydes etc.). Some of theprosthetic groups are designed to be coupled directly to a peptide or aprotein via formation of an amide linkage using an amine function of anamino acid residue (e.g. N-terminal α-NH₂ or internal ε-NH₂ of a lysine)or optionally via any other spacer containing an amino function. Inthese cases, the prosthetic groups are characterized by a carboxylicfunction (e.g. [¹⁸F]-FBA) usually activated as an active derivative(e.g. succinimidyl or nitrophenyl ester of the corresponding carboxylicacid).

All these radiolabelled prosthetic compounds are characterized bydifferent synthesis criteria such as the nature and ease of synthesis ofthe radiolabelling precursor, the effectiveness of the fluorinationstage, the total number of radiosynthesis stages, the time of synthesis,their overall radiochemical yield, the ease of purification, theireffectiveness in the conjugation reaction and the in vivo stability ofthe corresponding bioconjugates.

Moreover, the large scale production of these radiolabelled compounds isfaced with constraints related to the complete automation of theirsynthesis. In fact, a complete automated synthesis of thoseradiolabelled compounds will satisfy both the pharmaceutical standardsprocedures (GMP), as well as the radiological protection requirements.Therefore an ideal manufacturing procedure will be characterized by fewand easy synthesis and purifications steps.

Here below are shortly reported the synthesis of two of the prostheticgroups described in the literature:

-   -   the [¹⁸F]-SFB (N-succinimidyl 4-[¹⁸F]-fluorobenzoate ester):

Recently, EP2404903A1 described a three-step automated method forsynthesizing [¹⁸F]-SFB using microsynthesis technique:

Where:

“K222” corresponds to(4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane

“TSTU” corresponds to: (O—(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate.

Even if this automated radiosynthesis leads to reasonable radiochemicaldecay corrected yields (60%), it has several disadvantages. It is not soeasy to separate the labelled intermediate (fluoro benzoic ester) fromthe by product originated from the ammonium precursors. Moreover, thelarge number of steps and the necessity to purify each intermediate makethe automation of this process difficult even if it is necessary toreach the Good Manufacturing Practice conditions.

A further not insignificant drawback is the lipophilic character of[¹⁸F]-SFB reagent which makes difficult its “wet” conjugation towater-soluble amine-containing biomolecules and the purification step(HPLC or solid-phase extraction) of the resulting [¹⁸F]-labelledmolecular bioprobes.

-   -   The [¹⁸F]-SFS        (1-{4-[(2,2-dioxido-1,2-oxathiolan-3-yl)carbonyl]phenyl}-4-fluoro-1-oxobutane-2-sulfonic        acid):

WO2011/018467A1 relates to polysultone derivatives used as precursors toradiolabelled macromolecules for medical applications especially innuclear imaging and therapy.

-   -   Non radiolabelled Precursors

-   -   (Radio)labelled intermediates (prosthetic groups)

-   -   Where Ri is e.g. a radioelement or an NIR (Near Infra Red) agent    -   Where M is e.g. a generic cation    -   Labelled (bio)conjugates

The main advantage of these polysultones is that they can be coupledwith a large sort of (bio)molecules. In fact, they can be used not onlyfor the coupling with amino functions but also with thiol and hydroxylfunctions. The fast hydrolysis of these polysultone derivativesrepresents the main disadvantage of those prosthetic groups.

Objectives and Technical Problem

The present invention aims to combine the advantages of the abovementioned prosthetic groups avoiding their disadvantages by satisfyingat least one of the following objectives:

i) To provide novel compounds, the synthesis of which isstraightforward, easy, time-unconsuming, cheap and automatable, andwhich are capable of constituting non-labelled reactive precursors ofsynthons (prosthetic groups) for (radio)labelling complex andfragile—especially water-soluble and more especiallyamine-containing-(bio)molecules, in order to produce economical andeffective (radio)pharmaceuticals.

ii) To provide novel prosthetic groups, the synthesis of which isstraightforward, easy, time-unconsuming, cheap and automatable, andwhich are capable of constituting (radio)labelled intermediates forlabelling complex and fragile—especially water-soluble and moreespecially amine-containing—(bio)molecules in order to produceeconomical and effective (radio)pharmaceuticals.

iii) To provide novel labelled conjugates of complex andfragile—especially water-soluble and more especiallyamine-containing—(bio)molecules useful as effective(radio)pharmaceuticals, and obtained from prosthetic compounds, thesynthesis of which is straightforward, easy, time-unconsuming, cheap andautomatable.

iv) To provide a novel, easy, time-unconsuming, cheap and automatablemethod for synthesis of the precursor of the prosthetic group(non-labelled reactive precursors) as referred to in (i) supra.

v) To provide a novel, easy, time-unconsuming, cheap and automatablemethod for synthesis of the novel labelled prosthetic intermediate(labelled reactive precursors) as referred to in (ii) supra.

vi) To provide a novel method for obtaining novel (radio)labelledconjugates as referred to in (iii) supra, by labelling complex andfragile—especially water-soluble and more especiallyamine-containing—(bio)molecules, via coupling with a (radio)labelledprosthetic compound (labelled reactive intermediate) as referred to in(ii) supra, in order to produce economical and effective(radio)pharmaceuticals; said method offering at least one of thefollowing advantages:

-   -   a reduction in synthesis stages,    -   an increase in (radio)chemical yields at room temperatures (RT)        and within very short reaction time,    -   ease of separation of the intermediate and final product,    -   no production of by-products,    -   suitability for (radio)labelling different (bio)molecules,    -   possibility of carrying out the coupling of the (radio)labelled        intermediate with the (bio)molecule in water.

vii) To provide novel drugs and/or effective and economical(radio)tracers, from these new compounds as referred to in (i) & (ii)supra and conjugates as referred to in (iii) supra and this new(bio)molecule (radio)labelling method as referred to in (vi) supra.

viii) To provide a novel use of these new compounds as referred to in(i) & (ii) supra for (radio)labelling (bio)molecules with anucleophilicradionuclide or NIR imaging agent.

ix) To provide a novel use of these new compounds as referred to in (i)& (ii) supra, for imparting water-solubility to (bio)molecules bearingat least one nucleophilic group.

BRIEF DESCRIPTION OF THE INVENTION

These objectives, among others, are satisfied by the followinginvention.

In a first aspect, this invention relates to new compounds of formula:

(I)

in which:

-   -   the R⁰ bi-functional group is a spacer, preferably but not        exclusively chosen among the following radicals:

-   -   the R¹ monovalent hydrocarbon group is an activating group of        the oxygen atom of the ester function, R¹ preferably        corresponding to a succinimidyl ester, a benzotriazole ester, a        paranitrophenyl ester or a protecting labile (preferably        acid-labile) function or hydrogen;    -   the R² monovalent group corresponds to a hydrogen, a metallic        cation, an alkyl, a cyclo-alkyl, an aryl, an arylalkyl, an        alkylaryl, an acyl, an alkenyl, an alkynyl radical or a        combination of these radicals; hydrogen being preferred;    -   the R^(2′) monovalent group, corresponds to an hydrogen or an        alkyl, a cyclo-alkyl, an aryl, an arylalkyl, an alkylaryl, an        acyl, an alkenyl, an alkynyl radical or a combination of these        radicals; hydrogen being preferred;    -   the R³ bi-functional group corresponds to a hydrocarbon moiety,        preferably to a radical —(CR⁴R⁵)_(n)—, wherein R⁴, R⁵ represents        individually hydrogen or an alkyl, a cyclo-alkyl, an aryl, an        arylalkyl, an alkylaryl, an acyl, an alkenyl, an alkynyl radical        or a combination of these radicals; preferably hydrogen; n is        preferably but not exclusively an integer between 1 and 3;    -   R* is a nucleophilic radical preferably containing at least one        (radio)nuclide, preferably but not exclusively selected from the        list fluorine-18, bromine-76, iodine-123, iodine-124, iodine-131        or characterized by NIR properties.

The compounds according to the invention are:

-   -   Preferably compounds of general formula:

wherein

-   -   R¹, R²; R^(2′); R⁴; R⁵ and R* are as defined above.    -   and more preferably compounds of general formula:

wherein

-   -   R²; R^(2′); R⁴; R⁵ and R* are as defined above, R²; R^(2′); R⁴;        R⁵ preferably corresponding to hydrogen.

An example of these new compounds which is simply, economically andrapidly obtainable through an optimised multi-step protocol, is amono-fluorinated prosthetic compound of formula (III) wherein R* isfluorine, said compound being able as its analogues, to radiolabelcomplex and fragile amine-containing (bio)molecules.

The sulfonate function of these compounds (I); (II); (III) not only makethem soluble in water but also allows an easy separation from theirapolar precursors.

In a second aspect, the invention also pertains to the precursors offormula:

wherein

-   -   R⁰; R³ are as defined above;    -   R¹⁰ is a protective monovalent group that avoid any side        reaction on the carboxylic function and that makes possible the        reaction of the precursor (Ip) with a R* bearing nucleophilic        compound and corresponds to an alkyl, a cyclo-alkyl or possibly        a hydrogen or to R¹ as above defined, in case where R¹ permits        the reaction of the precursor (Ip) with a R* bearing        nucleophilic compound;    -   the functional units of formula COOR¹⁰ and

being nucleophile-reactive, the nucleophilic reactivity of thesefunctional units being different, COOR¹⁰'s nucleophilic reactivity beingpreferably less than the nucleophilic reactivity of

Preferably, these precursors are those of formula:

wherein

-   -   R¹⁰ is a monovalent group as defined above;    -   the functional units of formula COOR¹⁰ and

being nucleophile-reactive, the nucleophilic reactivity of thesefunctional units being different, R¹⁰'s nucleophilic reactivity beingpreferably less than the nucleophilic reactivity of

The sultone and the COOR¹⁰ nucleophile-reactive functional moietieswithin the same (e.g. benzenic) scaffold makes it possible to reach therequired chemical orthogonality between these moieties through asimple/easy/time-unconsuming protecting group strategy fully compatiblewith the requirements of automation.

In a third aspect, a subject of the invention is a method for synthesisof the precursors (Ip) or (IIp) as above defined, characterized in thatit essentially consists in:

i) implementing a structure, containing an ester and at least a secondreactive function, of formula:

wherein

-   -   the R⁰ bi-functional group is as defined supra;    -   X is the second reactive function suitable to act as a leaving        group during a nucleophilic substitution. X is preferably but        not exclusively an alkoxide function, an halogen as chlorine,        bromine, iodine or a triflate, a tosylate or a mesylate.    -   the R^(10′) monovalent group corresponds preferably but not        exclusively to an alkyl or a cyclo-alkyl, one of the ester        functions OR^(10′) or an hydrogen

ii) making the structure (IV) react with:

-   -   at least one sultone—advantageously a butane sultone, propane        sultone and/or an ethane sultone—, the sultone being preferably        firstly metalated by means of a deprotonating agent, preferably        n-butyl-lithium, then acylated;    -   and with a protecting reagent capable of substituting the        R^(10′) function in (IV) by a protecting labile, preferably        acid-labile, function R^(10″).

In a fourth aspect, a subject of the invention is a method for thesynthesis of the compounds (I); (II) & (III) derived from the sultoneprecursors (Ip), characterized in that it comprises the followingstages:

a. utilization or synthesis of a precursor (Ip), or obtained by themethod as above defined;

b. opening of the sultone of the precursor with a R* bearingnucleophilic radical leading to the formation of a sulphonate, beingpreferably carried out either in a polar protic typically as alcoholssolvent or in the presence of a polar aprotic solvent which morepreferably contains some water in an amount—given in an increasing orderof preference and in % w/w—of less than or equal to 15; 10; 8; 6; 5;comprised between 1-4; 2-4;

c. deprotection of the protected labile ester function;

d. activation of the carboxylic function obtained as mentioned on pointc. by grafting of a R¹ monovalent group as defined supra;

e. recovery of the sulphonated nucleophilic-reactive compound obtainedin stage d.

In a fifth aspect, the invention relates to the conjugates made from thecompounds (I); (II) & (III) and at least one active (bio)molecule aswell as to the process for making these conjugates.

In a sixth aspect, the invention relates to a drug comprising at leastone compound according to the invention or obtained by one of themethods according to the invention.

In a seventh aspect, the invention relates to the use of the compoundsaccording to the invention or obtained by the method according to theinvention for (radio)labelling (bio)molecules with nucleophilicradionuclides or Near Infra Red (NIR) agents,

or for imparting water-solubility to (bio)molecules bearing at least onenucleophilic group.

The main advantages of the invention are the following:

-   -   Use of the same approach for (radio)labelling irrespective of        the radionuclide, NIR agent or labelling agent;    -   Obtaining high (radio)chemical yields in mild conditions (at        room temperatures and within very short reaction time);    -   Possibility of easily separate the starting precursor (apolar)        from the product formed (polar);    -   No production of by-products;    -   Possibility of automation of the complete synthesis procedure;    -   Suitability for labelling numerous and various        (bio)macromolecules;    -   Possibility to conjugate with the (bio)molecules in aqueous        conditions;    -   Simplicity;    -   Economy;    -   Access to novel compounds opening up multiple therapeutic and        diagnostic developments.

DETAILED DESCRIPTION OF THE INVENTION Definitions

According to the terminology of this text, the following non limitativedefinitions have to be taken into consideration:

-   -   Every singular designates a plural and reciprocally.    -   “(bio) molecules” or “biomolecules” refers notably to biological        macromolecules, such as peptides, proteins, antibodies,        oligonucleotides, nucleic acids, carbohydrates, lipids,        nanoparticles, biopolymers, and dendrimers.    -   “prosthetic” means a functionalised group or a functionalised        compound which is intended to or is conjugated (tightly bound)        with complex and fragile (bio)molecules, which could be a        nonpolypeptide structure and which could or not be required for        the activity of the (bio)molecule. This conjugate could be a        radiotracer wherein the prosthetic group carries the chosen        radionuclide.    -   In the formulae, notably those of the novel compounds (I); (II);        (III); (Ip); (IIp), reference is made to the following        definitions:

“alkyl” corresponds for example to a linear, branched or cyclicsaturated monovalent C1-C30 alkyl group, preferably C1-C20, and, evenmore preferentially C1-C10, optionally substituted, comprising or notcomprising heteroatoms. Examples of alkyl groups are in particularmethyl, ethyl, isopropyl, n-propyl, tert-butyl, isobutyl, n-butyl,n-pentyl, isoamyl and 1,1-dimethylpropyl.

“aryl” corresponds for example to one or more monocyclic or polycyclicand preferably monocyclic or bicyclic condensed or uncondensed aromaticmonovalent groups, having 6 to 18 carbon atoms. It must be understoodthat, within the framework of the invention, by polycyclic aromaticradical is meant a radical having two or more aromatic rings, condensed(orthocondensed or ortho- and pericondensed) with each other, i.e.having, in pairs, at least two carbon atoms in common. Said aromatichydrocarbon group (“aryl”) is optionally substituted for example by oneor more C₁-C₃ alkyls, one or more halogenated hydrocarbon radicals (e.g.CF₃), one or more alkoxy (e.g. CH₃O) or one or more hydrocarbon radicalscomprising one or more ketone units (e.g. CH₃CO—). By way of examples ofaryls, there can be mentioned the phenyl, naphthyl, anthryl andphenanthryl radicals.

“arylalkyl” corresponds for example to an alkyl group as defined above,substituted by one or more aryl groups on its hydrocarbon chain, thearyl group being as defined above. Examples of this are benzyl andtriphenylmethyl.

“alkylaryl” corresponds for example to monovalent alkyl, substituted orlinked to one or more monovalent aromatic groups, optionallysubstituted.

By “acyl” is meant an R⁰—CO— group where R₀ represents alkyl as definedabove; or an Ar—CO— group where Ar represents an aryl group as definedabove, or arylalkyl in which aryl and alkyl are as defined above and inwhich the aryl part is optionally substituted e.g. by alkyl.

By “cycloalkyl” is meant a mono- or polycyclic, preferably mono- orbicyclic, saturated hydrocarbon radical preferably having from 3 to 10carbon atoms, even better from 3 to 8. By polycyclic saturatedhydrocarbon radical is meant a radical having two or more cyclic ringsattached to each other by a bonds and/or condensed in pairs. Examples ofpolycyclic cycloalkyl groups are adamantane and norbornane. Examples ofmonocyclic cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl.

By “alkenyl” is meant e.g. a substituted or unsubstituted, linear orbranched, unsaturated hydrocarbon chain, having at least one olefinicdouble bond, and more preferably a single double bond. Preferably, thealkenyl group has 2 to 8 carbon atoms, even better 2 to 6. Thishydrocarbon chain optionally comprises at least one heteroatom such asO, N, S. Preferred examples of alkenyl groups are the allyl andhomoallyl groups.

By “alkynyl” is meant e.g. according to the invention, a substituted orunsubstituted, linear or branched, unsaturated hydrocarbon chain, havingat least one acetylenic triple bond, and more preferably a single triplebond. Preferably, the alkynyl group has 2 to 8 carbon atoms, even better2 to 6 carbon atoms. By way of example, there can be mentioned theacetylenyl group, as well as the propargyl group. This hydrocarbon chainoptionally comprises at least one heteroatom such as O, N, S.

Preferences

The Compounds and their Precursors According to the Invention:

-   -   Spacer R⁰ corresponds to

Compounds (I); (II); (III) are for example:

R* is preferably ¹⁸F

Precursor (Ip) corresponds to:

-   -   Spacer R⁰ corresponds to —CH₂C(O)—

Compounds (I); (II); (III) are for example:

R* is preferably ¹⁸F

Precursors (Ip) is:

-   -   Spacer R⁰ corresponds to

Compounds (I); (II); (III) are for example:

Precursors (Ip) corresponds e.g. to:

-   -   Spacer R⁰ corresponds to

Compounds (I); (II); (III) are for example:

Precursors (Ip) e.g. is:

Synthesis of Precursors (Ip)

These precursors can be synthesized from commercial products.

Thus, the method for synthesizing the precursors (Ip) essentiallyconsists in making react, preferably, a monoester of a dicarboxylicproduct, in particular an aromatic dicarboxylic product (e.g.terephthalic acid), the CO₂H moiety of which being protected through anacid-labile group namely tert-butyl ester, with a sultone metalated withn-BuLi and subsequently acylated. For example, this synthesis can besummarized on the scheme 1 as follows:

Scheme 1 Reagents and conditions: (a) tert-butyl2,2,2-trichloroacetamidate, CH₂Cl₂, 35° C., overnight; (b)1,3-propanesultone, n-BuLi, THF, −78° C., 3 h 30 then acetic acid, THF,−78° C. to rt, overall yield 51%.

Butane sultones and/or ethane sultones can be used instead of or inaddition to propane sultones:

Synthesis of the Compounds (I); (II); (III)

According to the invention, the method for obtaining the novel compounds(I); (II); (III) is preferably done from the precursors (Ip), (IIp)according to a three-stage synthetic scheme:

1. Introduction of the R* radical, for example fluorination of (IIp) bymeans of a R* (e.g. fluorine) bearing reagent (e.g KF/Kryptofix [K222]),to obtain efficiently the desired R* (e.g. fluoro)-sulfonated derivative(II);

The reaction solvent can be a polar aprotic solvent containing traces ofwater (e.g. 3%) or a protic solvent;

-   -   the polar aprotic solvent being preferably but not exclusively        selected among the followings: acetonitrile, dimethylsulfoxide        (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), toluene        or a mixture of them;    -   the protic solvent being preferably but not exclusively selected        among the alcohols, and advantageously among the group of        alcohols comprising—preferably but not exclusively composed of        MeOH, EtOH, i-PrOH, t-BuOH, Amyl alcohol, or a mixture of them;        [stage 1 corresponds to the steps a & b of the method for        obtaining the novel compounds (I); (II); (III) according to the        claims].

2. Removal of the R¹ corresponding to R^(10″) radical (e.g tert-butyl)of the R* (e.g.fluoro)-sulfonated derivative (II) by treatment, such ashydrolysis (e.g with a 25% TriFluroAcetic acid solution in CH₂Cl₂) orhydrogenolysis, depending on the protecting agent, to give a free (e.gbenzoic) acid (II) wherein R¹ corresponding to hydrogen.

[stage 2 corresponds to the step c of the method for obtaining the novelcompounds (I); (II); (III) according to the claims].

3. Finally:

i) Activation of the carboxylic function with an activating agent [e.g.N-HydroxySuccinimide (NHS); para-nitrophenyl] in a dry polar aproticsolvent (e.g CH₃CN);

ii) or reaction of R* (e.g.fluoro)-sulfonated free (e.g benzoic) acid(II) wherein R¹ corresponding to hydrogen, with a coupling reagent [e.gO—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU)]and a base [e.g. N,N-Diisopropylethylamine (DIEA) in a dry polar aproticsolvent [e.g. N-Methyl-2-pyrrolidone (NMP)]; led to the bioconjugatableactivated ester (III) in almost quantitative yield.

[stage 3 corresponds to the step d of the method for obtaining the novelcompounds (I); (II); (III) according to the claims].

The so obtained sulphonated nucleophilic-reactive compounds (II) or(III) are collected. [step e of the method for obtaining the novelcompounds (I); (II); (III) according to the claims].

These details of implementation make it possible to increase theelectrophilicity of the carbonyl function and to couple withnucleophilic functions such as amino acids, peptides, in mildconditions.

According to a remarkable and particular embodiment of the method forobtaining the novel compounds (I); (II); (III) according to theinvention, the R* (e.g. fluorine) bearing reagent is adsorbed onto anelutable support and is afterwards eluted into a reactor wherein itreacts with the precursor (Ip).

The R* (e.g. fluorine) is eluted into the reactor by means of an eluentsolution comprising at least one polar aprotic or protic solvent and atleast one phase transfer agent.

The possible polar aprotic solvent of the eluent solution is preferablyselected in the group comprising—even better composed of—CH₃CN, DMF,DMSO, THF, toluene, mixture CH₃CN/DMF or DMSO/water.

The possible protic solvent of the eluent solution is preferablyselected in the group comprising—ideally composed of—water MeOH, EtOH,i-PrOH, t-BuOH, Amyl alcohol or a mixture of them.

The phase transfer agent is e.g. chosen between a quaternary amine (e.g.N(C₄H₉)⁺ B) or a compound of general formula Kriptand/MxBy, whereinKriptand is a molecule suitable for a stable coordination of the metal Mand were M is an alkaline metal, alkaline earth metal and in both casesB is a counter ion as (but not only) carbonate, bicarbonate, oxalate.More preferably but not exclusively, said phase transfer agent beingselected in the group comprising—ideally composed of—: K222/Na2CO3;K222/K2CO3; K222/Cs2CO3; K222/Rb2CO3; TBAHCO3; K222/K2C2O4; K222/NaHCO3;K222/KHCO3; K222/RbHCO3; K222/CsHCO3 where K222 corresponds to thekriptand (4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane)and mixtures thereof.

After removal of the eluent solvents, the precursor (IIp) is added intothe reactor as solution of a protic or aprotic solvent.

The possible polar aprotic solvent used to make the (IIp) solution ispreferably selected in the group comprising—even better composedof—CH₃CN, DMF, DMSO, THF, toluene, mixture CH₃CN/DMF or DMSO/water.

The possible protic solvent used to make the (IIp) solution ispreferably selected in the group comprising—ideally composed of—waterMeOH, EtOH, i-PrOH, t-BuOH, Amyl alcohol or a mixture of them.

Into the reactor R* (e.g. fluorine) reacts with the precursor (IIp).

In a preferred case, the reaction between the R* (e.g. fluorine) bearingreagent and the precursor (IIp) is done in preferentially less than 15minutes, at a temperature greater or equal to the room temperature,comprised between in an increasing order of preference: 30 and 150° C.;40 and 120° C.; 50 and 110° C.; 60 and 100° C.; 80 and 100° C.

This fluorinated intermediate is then hydrolized in order to remove theprotecting group of the carboxyl function by means either of acidic orbasic hydrolysis or hydrogenation. The resulting carboxylic acidderivative is then activated as reactive ester derivative by means ofany coupling agent such as TSTU or the combination DCC/NHS. At thisstep, the active ester can either be used for direct coupling withvarious (bio)molecules or further purified if necessary.

Here below is reported a general scheme for the labelling of theprecursor (IIp) with fluorine. The differences between the labellingwith F-19 and the radiolabelling with F-18 are reported into thecaption.

Scheme 2 Reagents and conditions:Labelling using F-19: (a) KF, Kryptofix[K222], CH₃CN—H₂O (98:2, v/v), RTRP-HPLC purification, 63%; (b) TFA, CH₂Cl₂, RT, 1 h, quantitative yield;c) DCC, NHS, CH₃CN RT, 1 h or TSTU, DIEA, NMP, 30 min, quantitativeyield.Labelling using F-18(a) see Table 1, entries 6-8; (b) 4.0 M aq. HCl, 80°C., 5 min; (c) TSTU, DIEA, CH₃CN, 50° C., 5 min.

Conjugation of the (Radio)Labelled Compounds (I); (II); (III) with(Bio)Molecule

The novel nucleophilic(amine)-reactive prosthetic compounds (I); (II);(III) are useful for (radio)labelling and imparting water-solubility ofmolecular architectures, e.g. of fragile and hydrophobic molecules.

Such a conjugation can be for instance implemented with anamine-reactive (bio)molecule through an amidification reaction betweenthe compounds (I); (II); (III), especially the NHS ester of [¹⁸F]-(III)and a primary amino group present onto the amine-reactive (bio)molecule.

By way of example, this latter can be a far-red fluorescent marker:amine derived from the pentamethine cyanine 5.5 (Cy 5.5) core or apolypeptide containing lots of hydrophobic lateral chains.

The conjugation results from an amidification reaction between the NHSester of [¹⁸F]-(III) and a primary amino group present on the cyaninescaffold.

This conjugated amino-fluorophore is obtained through a two-stepsreaction sequence (i.e., amidification followed by removal of thephthalimide protecting group) from a cyanine phthalimide-acidderivative.

The amidification comprises an amidolysis of active esters involving abase e.g. a tertiary amine such as DIEA in a dry polar aprotic solventsuch as N-Methyl-2-Pyrrolidone (NMP).

The final fluorinated/sulfonated Cy 5.5 derivative is collected in apure form after purification achieved e.g. by RP-HPLC.

Moreover the originally water insoluble precursor Cy 5.5 becames solublein aqueous buffers thanks to the conjugation with the very polar andhydrophilic sulfonate derivative. This fact confirms that the presentinvention enables to increase the hydrophilic character of the(bio)molecules conjugated therewith.

CONCLUSION

The new chemical path opened by the invention which consists inpre-introduction of a marker R*, e.g. Fluorine-18, through an unusualnucleophile-induced ring-opening reaction of the sultone (e.g.1,3-propanesultone) moiety of an heterobifunctional precursor. Thisnucleophilic substitution leads to the disclosure of a free sulfonicacid moiety which is greatly beneficial, since it accelerates andfacilitates the purification of the resulting R*-conjugates bydrastically modifying their intrinsic hydrophilic character. Thus, these(radio)synthesis procedures can be rapidly and easily implemented andautomated. They are readily reproducible and give very satisfactoryyields within very short reaction times (around 1 minute). Theseadded-values make it clear that the novel prosthetic compounds andprecursors according to the invention, represent a viable alternativenotably to [¹⁸F]-SFB. Furthermore, the mild reaction conditionsassociated with the chemistry of the active ester (e.g. NHS activeester) enables to achieve the R* [especially ¹⁸F]-(radio)labelling ofpeptides or highly-functionalised and fragile fluorescent markers.Moreover, this technology when applied to an active targeting moleculespermit to obtain a R*[especially F-18]-(radio)labelled compound suitablefor medical applications such as nuclear imaging, optical imaging andeven the combination of both of them in the case of [¹⁸F]-PET/NIRF dualmodality agents.

EXAMPLES

Unless otherwise noted, all other commercially available reagents andsolvents are used without further purification. CH₂Cl₂ and CH₃CN aredried through distillation over P₂O₅ and CaH₂ respectively. AnhydrousTetrahydrofuran (THF) is obtained through drying over Na+/benzophenone.Anhydrous Dimethylformamide (DMF) is obtained from Carlo Erba-SdS orFisher Scientific. Peptide synthesis-grade N-Methyl-2-pyrrolidone (NMP)is purchased from Carlo Erba-SdS. Bovine serum albumin (BSA) protein andKryptofix[K222] (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane) are purchased from Sigma-Aldrich. TLC are carried out onMerck DC Kieselgel 60 F-254 aluminium sheets. The spots are visualisedby illumination with UV lamp (λ=254 nm) and/or staining with KMnO₄solution. Flash column chromatography purifications are performed onGeduran® Si 60 silica gel (40-63 μm) or (63-200 μm for cynaninederivatives) from Merck. Cyanine amino carboxamide 6 is preparedaccording to a synthetic scheme described in the ESI file). Thesynthesis of dodecapeptide (N^(α)-Ac-lysine-terminated of formula:AcKGRANLRILARY is carried out on an Applied Biosystems 433A peptidesynthesizer using the standard Fmoc/tBu chemistry16 and the Wang resin(Iris Biotech, loading 0.9 mmol/g) on a scale of 0.25 mmol. TheHPLC-gradient grade acetonitrile (CH₃CN) and methanol (CH₃OH) areobtained from VWR. Phosphate buffered saline (PBS, 100 mM phosphate+150mM NaCl, pH 7.5) and aq. mobile-phases for HPLC are prepared using waterpurified with a Milli-Q system (purified to 18.2 MΩ·cm).Triethylammonium bicarbonate (TEAB, 1.0 M) buffer is prepared fromdistilled triethylamine and CO2 gas

Instruments and Methods.

NMR spectra (¹H, ¹³C and ¹⁹F) are recorded on a Bruker DPX 300spectrometer (Bruker, Wissembourg, France) or with a Bruker AC 200.Chemical shifts are reported in parts per million (ppm) downfield fromresidual solvent peaks: CDCl₃ (δ_(H)=7.26, δ_(C)=77.16) or CD₃OD=3.31,δ_(C)=49.0)¹⁷ and coupling constants are reported as Hertz (Hz).Splitting patterns are designated as singlet (s), doublet (d), doubledoublet (dd), double double doublet (ddd) and triplet (t). Splittingpatterns that could not be interpreted or easily visualised aredesignated as multiplet (m). ¹³C substitutions are determined with JMODexperiments, differentiating signals of methyl and methine carbonspointing “up” (+) from methylene and quaternary carbons pointing “down”(−).

The elemental analyses are carried out with a Flash 2000 OrganicElemental Analyzer (Thermo Scientific). Analytical HPLC is performed ona Thermo Scientific Surveyor Plus instrument equipped with a PDAdetector. Semi-preparative HPLC is performed on a Thermo ScientificSPECTRASYSTEM liquid chromatography system (P4000) equipped with aUV-visible 2000 detector. Mass spectra are obtained with a Finnigan LCQAdvantage MAX (ion trap) apparatus equipped with an electrospray (ESI)source. UV-visible absorption spectra are obtained on a Varian Cary 50scan spectrophotometer by using a rectangular quartz cell (Varian,standard cell, Open Top, 10×10 mm, 3.5 mL).

Fluorescence spectroscopic studies (emission/excitation spectra) areperformed on a Varian Cary Eclipse spectrophotometer with a semi-microquartz fluorescence cell (Hellma, 104F-QS, 10×4 mm, 1400 μL). Fordetails related to the determination of real time quantum yields, see“Electrospray ionization”, the article on Wikipedia.

Fluoride-18 is produced by the ¹⁸O[p,n]¹⁸F nuclear reaction using a GEMedical Systems PETtrace cyclotron [18 MeV proton beam] (AdvancedAccelerator Applications, Saint-Genis-Pouilly, France) and ¹⁸O-enrichedwater purchased from Marshall Isotopes Ltd. (98%, Tel Aviv, Israel).

Solid-phase extraction (SPE) cartridges (SepPak QMA Light, Oasis HLB andCM) are obtained from ABX advanced biochemical compounds (Radeburg,Germany) and Waters (Guyancourt, France). The HLB cartridges are alwayspre-conditioned with ethanol (5 mL), water (5 mL) and dried with air.

Radiosyntheses are performed on a TRACERlab MX (GE Medical Systems, Buc,France) automated synthesis unit in a shielded hot cell (8 cm lead,Comecer, Castel Bolognese, Italy).

A flow-count radio-HPLC detector system from Bioscan is used only forHPLC analyses (performed on a Dionex UltiMate® 3000 LC system) ofreactions involving ^(18F).

HPLC Separations.

Several chromatographic systems are used for the analytical experimentsand the purification steps: System A: RP-HPLC (Thermo Hypersil GOLD C18column, 5 μm, 4.6×100 mm) with CH₃CN and 0.1% aq. trifluoroacetic acid(aq. TFA, 0.1%, v/v, pH 2.0) as eluents [100% TFA (5 min), lineargradient from 0% to 80% (40 min) of CH3CN] at a flow rate of 1.0 mLmin-1. Dual UV detection is achieved at 254 and 265 nm. System B:RP-HPLC (Thermo Hypersil GOLD C18 column, 5 μm, 2.1×100 mm) with CH3CNand 0.1% aq. trifluoroacetic acid (aq. TFA, 0.1%, v/v, pH 2.0) aseluents [80% TFA (5 min), linear gradient from 20% to 40% (5 min) and40% to 100% (50 min) of CH₃CN] at a flow rate of 0.25 mL min-1. UV-visdetection with the “Max Plot” (i.e., chromatogram at absorbance maximumfor each compound) mode (220-798 nm). System C: RP-HPLC (Thermo HypersilGOLD C18 column, 5 μm, 10×250 mm) with CH₃CN and 0.1% aq. TFA as eluents[100% TFA (5 min), linear gradient from 0% to 20% (10 min), 20% to 45%(25 min), 45% to 65% (10 min) and 65% to 100% (5 min) of CH3CN] at aflow rate of 5.0 mL min-1. Dual UV detection is achieved at 270 and 300nm. System D: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 μm, 21.2×250mm) with CH3CN and 0.1% aq. TFA as eluents [100% TFA (5 min), lineargradient from 0% to 10% (5 min), 10% to 30% (20 mM), 30% to 50% (10 min)and 50% to 100% (15 min) of CH3CN] at a flow rate of 15.0 mL mM-1. DualUV detection is achieved at 270 and 300 nm. System E: RP-HPLC (VarianKromasil C18 column, 10 μm, 21.2×250) with CH₃CN and aq. TEAB (50 mM, pH7.5) as eluents [100% TEAB (5 mM), linear gradient from 0% to 30% (10min) and 30% to 100% (70 min) of CH3CN] at a flow rate of 20 mL min-1.Dual visible detection is achieved at 625 and 680 nm. System F: system Cwith the following gradient [90% TFA (5 mM), linear gradient from 10% to100% (36 min) of CH₃CN] at a flow rate of 4.0 mL min-1. Dual visibledetection is achieved at 625 and 680 nm. System G: system A with thefollowing gradient [80% TFA (5 min), linear gradient from 20% to 100%(40 min) of CH₃CN] at a flow rate of 1.0 mL min-1. Dual UV detection isachieved at 220 and 260 nm. System H: RP-HPLC (Theimo Hypersil GOLD C18column, 5 μm, 10×100 mm) with CH3CN and 0.1% aq. TFA as eluents [100%TFA (5 min), linear gradient from 0% to 80% (40 mM) and 80% to 100% (5min) of CH₃CN] at a flow rate of 4.0 mL min-1. Dual UV detection isachieved at 227 and 261 nm. System I: system A with the followinggradient [100% TFA (3.8 mM), linear gradient from 0% to 44% (16.9 min)and 44% to 100% (3.8 min) of CH₃CN] at a flow rate of 1.3 mL mM-1. DualUV detection is achieved at 254 and 265 nm.

Example 1: Sultone-Benzoic Acid, Tert-Butylester (2)

Scheme 1 Reagents and conditions: (a) tert-butyl2,2,2-trichloroacetamidate, Cf₂Cl₂, 35° C., overnight; (b)1,3-propanesultone, n-Buli, THF, −78° C., 3 h 30 then acetic acid, THF,−78° C. to RT, overall yield 51%.

(a) Esterification: To a stirred solution of mono-methyl terephthalate(500 mg, 2.78 mmol, 1 equiv.) in dry CH₂Cl₂ (15 mL), under an argonatmosphere, is added tert-butyl 2,2,2-trichloroacetimidate (1.25 g, 5.55mmol, 2 equiv.). The resulting reaction mixture is stirred at 35° C.overnight. Then, the crude mixture is filtrated to remove the remainingunreacted mono-methyl terephthalate acid and is then purified byflash-chromatography on a silica gel column using a mixture ofcyclohexane-ethyl acetate (9:1, v/v) as the mobile phase. After removalof the solvent under vacuum, the resulting pure solid is directly usedin the next step. TLC analysis: Rf 0.73 (cyclohexane-EtOAc, 3:7, v/v).

(b) Acylation of 1,3-propanesultone: To a stirred solution of commercial1,3-propanesultone (720 mg, 5.89 mmol, 2.1 equiv.) in dry THF (10 mL),under an argon atmosphere, at −78° C., is added dropwise n-BuLi (2.0 Min hexane, 3 mL, 6 mmol, 2.2 equiv.). After 1 h of stirring at −78° C.,a solution of the previously isolated tert-butyl methyl diester (videsupra) in dry THF (10 mL) is added dropwise to the previous vigorouslystirred mixture. The resulting reaction mixture is stirred at −78° C.for 2 h 30, then kept at −78° C., and quenched by adding 1 mL of glacialacetic acid dissolved in dry THF (3 mL). Thereafter, the reactionmixture is slowly warmed up to RT then diluted with brine (20 mL) andCf₂Cl₂ (50 mL). The product is extracted from the aq. Phase with CH₂Cl₂(30 mL). The combined organic layers are dried over anhydrous MgSO₄,filtered and then concentrated under reduced pressure. The resultingcrude product is then purified by flash-chromatography on a silica gelcolumn using a mixture of cyclohexane-EtOAc (gradient from 9:1 to 7:3,v/v) as the mobile phase. The desired product 2 is isolated as a whitepasty solid (458 mg, overall yield for the two steps 51%). TLC analysis:Rf 0.33 (cyclohexane-ethyl acetate, 3:7, v/v); HPLC (system A): Rt=31.4min (purity 97%).

Example 2: Mono-Fluoro-Sulfonated Tert-Butyl Ester (3)

Example 2.1: Synthesis of a Non-Radioactive [¹⁹F]Mono-Fluoro-SulfonatedTert-Butyl Ester (3)

To a stirred solution of Kryptofix[K222] (76.2 mg, 0.20 mmol, 3.3equiv.) and KF (10.7 mg, 0.184 mmol, 3 equiv.) in a mixture of dry CH₃CN(1 mL) and deionised water (20 μL), is added sultone 2 (20 mg, 0.061mmol, 1 equiv.). The resulting reaction mixture is stirred at roomtemperature and its completion is checked by analytical RP-HPLC (systemA). Thereafter, the crude product is purified by semi-preparativeRP-HPLC (system C). The product-containing fractions are lyophilised togive the desired sulfonic acid derivative 3 as a white amorphous powder(13.4 mg, yield 63%).

Example 2.2: Synthesis of Different Radioactive[¹⁸F]-Mono-Fluoro-Sulfonated Tert-Butyl Esters (3)

Radiosynthesis and subsequent purification are performed using a GeneralElectric TRACERlab MX device. The mono-sultone precursor 2 was engagedwith [¹⁸F]-fluoride at 90° C. for 10 min. Different compositions of theeluent solution used to transfer [¹⁸F]-fluoride to the reaction vialwere tested. The compositions and the results are summarised in Table 1.

TABLE 1 Selected reaction conditions for the preparation of [^(18F)]-3from the mono-sultone precursor 2. % conversion rate phase (radio- entrysolvent^(a) transfer agent^(b) HPLC)^(c) 1 CH₃CN K₂CO₃/K222 3 2 CH₃CNCs₂CO₃/K222 12 3 CH₃CN + K₂CO₃/K222 6.5 3% H₂O 4 CH₃CN + Cs₂CO₃/K222 323% H₂O 5 CH₃CN + Cs₂CO₃/K222 23 6% H₂O 6 t-BuOH Cs₂CO₃/K222 86 7 AmylCs₂CO₃/K222 70 alcohol 8 i-PrOH Cs₂CO₃/K222 90 ^(a)[¹⁸F]-labelling wascarried out in 1.0 mL of the respective solvent (mixture) for 10 min at90° C. except for entries 4-6 (1.6 mL). ^(b)With 1.8 equiv ofKryptofix[K222]. ^(c)Ratio between product [¹⁸F]-3 and free Fluorine-18in the crude reaction mixture.

With respect to acetonitrile as reaction solvent, the use of Cs₂CO₃instead of K₂CO₃ allowed to obtain better results but the conversionrates were still lower than 15% (entry 2). The presence of traces ofwater in the reaction solvent results in a slight increase of theconversion rate that however remains low (under 30%). Theradiofluorination of cyclic sulfonate ester 2 performed in differentsolvent mixtures named Isopropyl alcohol; amyl alcohol; tert butylalcohol, makes it possible to produce the [¹⁸F]-fluorinated tert-butylester [¹⁸F]-3 in good radiochemical yields and purity with respect toall previously performed reactions (entry 6-8).

Example 3: Mono-Fluoro-Sulfonated Benzoic Acid (4)

Example 3.1: Synthesis of a Non-Radioactive [¹⁹F] Mono-Fluoro-SulfonatedBenzoic Acid (4)

To a stirred solution of tert-butyl ester 3 (13.5 mg, 0.04 mmol) inCH2Cl₂ (1 mL), is added a solution of TFA in CH₂Cl₂ (1 mL, 1:1, v/v).The resulting reaction mixture is vigorously stirred at RT for 1 h.Completion of the reaction is checked by analytical RP-HPLC (system A).Then, the reaction mixture is evaporated under reduced pressured andco-evaporated three times with toluene (3×20 mL) to give the desiredproduct 4′ as a white solid (11.2 mg, quantitative yield).

Example 3.2: Synthesis of a Radioactive [¹⁸F] Mono-Fluoro-SulfonatedBenzoic Acid (4)

Following the [¹⁸F]-radiolabelling and purification steps, thetert-butyl ester of [¹⁸F]-3 is removed by treatment with 4.0 M aq. HClinstead of TFA (used for the synthesis of the corresponding[¹⁹F]-derivative). The reaction is performed at 80° C. for 5 min and theresulting free benzoic acid [¹⁸F]-4 was purified by solid-phaseextraction (SPE) using an Oasis® HLB cartridge.

Example 4: [F]-Prosthetic Group (1)

Example 4.1: Synthesis of a Non-Radioactive [¹⁹F]-Prosthetic Group (1)

Benzoic acid 4 (48 mg, 0.165 mmol) is dissolved in peptidesynthesis-grade NMP (1.5 mL). TSTU (49.8 mg, 0.165 mmol, 1 equiv.) andDIEA (165 μL of a 2.0 M solution in NMP, 0.33 mmol, 2 equiv.) aresequentially added and the resulting reaction mixture is stirred at roomtemperature for 30 min. The reaction is checked for completion by ESImass spectrometry. The crude NHS ester is used in the next amidificationreactions without prior purification-isolation.

Example 4.2: Synthesis of a Radioactive [¹⁸F]-Prosthetic Group (1)

Final elution of the acidic intermediate from the HLB cartridge withCH₃CN-DIEA (9:1, v/v) allows the recovery of the correspondingcarboxylate anion which subsequently reacts with a solution of TSTU inCH₃CN at 50° C. for 5 min, to provide the corresponding NHS ester. Thus,3-4 GBq of the targeted [¹⁸F]-labelled prosthetic compound [¹⁸F]-1 areobtained within 72 min, starting from 10-15 GBq of [¹⁸F]-fluoride(35-45% average decay-corrected radiochemical yield for n=15 and 80-95%radiochemical purity).

Example 4.3: Automated Synthesis of Radioactive [¹⁸F]-Prosthetic Group(1)

A multistep synthesis of this novel prosthetic compound is performed onan automated synthesizer General Electric TRACERlab MX equipped withstandard FDG cassettes. A new Excel® sequence, defining every step ofthe synthetic procedure, is also developed to control the module via acomputer. The FDG cassette is composed of three manifolds where solventsand reagents are charged: first manifold (position 1 to 5), secondmanifold (position 6 to 10) and third manifold (position 11 to 15). TheC18 and alumina cartridges are removed and the water bag (250 mL) istransferred from position 7 to 13. A vial of CH₃CN (7 mL) and onecontaining a solution of TSTU in CH₃CN are respectively placed inpositions 3 and 5. All the reactions take place in a single reactorwhich is cleaned with HCl and deionised water between purification andgeneration of the active ester. Appropriate detectors permit to followthe radioactivity during the synthesis, on the QMA and HLB cartridges,the reactor and the waste bottle. A Dose Calibrator is used to measureradioactivity into the final recovery vial. Following delivery of[¹⁸F]-fluoride to the synthesizer module, the radioactivity is isolatedon a QMA cartridge, allowing recovery of [¹⁸O]-H2O. The [18F]-fluorideis eluted with a mixed solution of Kryptofix[K222] (20.8 mg) in CH₃CN(400 μL) and of Cs₂CO₃ (9.8 mg) in deionised water (200 μL), andtransferred to the reaction vial. After azeotropic evaporation of waterwith CH₃CN (3×1 mL, 95° C., with a stream of N2 gas), sultone-benzoicacid, tert-butyl ester 2 (3.5 mg) in iPrOH (1 mL) is added. Theradiolabelling step is conducted into the reaction vial, at 90° C.during 10 min. After cooling, the reaction mixture is diluted with waterand loaded onto an Oasis® HLB cartridge. The reaction vial and cartridgeare washed with water, then the [¹⁸F]-sulfonated tert-butyl ester[18F]-3 is eluted with an aq. solution of CH₃CN (H₂O—CH₃CN, 75:25, v/v,3 mL) and transferred back to the reactor. The tert-butyl ester is thenremoved by treatment with 4.0 M aq. HCl (2 mL) at 80° C. for 5 min whilethe HLB cartridge is cleaned with CH₃CN (3 mL) and finally rinsed withwater (30 mL). After cooling, the reaction mixture is diluted with waterand the [¹⁸F]-sulfonated benzoic acid [¹⁸F]-4 is trapped onto the Oasis®HLB cartridge. The reaction vial and cartridge are washed with water,then [¹⁸F]-sulfonated benzoic acid [¹⁸F]-4 is eluted with a 10% solutionof DIFA in CH₃CN (2 mL) to the reactor. To the formed carboxylate anion,is then added a solution of TSTU in CH₃CN from an external line andactivation is performed at 50° C. for 5 min. The reaction mixture isthen transferred to the final vial. The reactor is rinsed with CH₃CN (2mL) and the solution transferred to the final vial. The activity ismeasured with the Dose Calibrator. [¹⁸F]-radiolabelling reagent [¹⁸F]-1is obtained within 75 min with a moderate 20-30% decay-correctedradiochemical yield (average value from n=10 preparations) and with a95% radiochemical purity. HPLC (system I): tR=12.1 min.

Example 5: Fluoro-Monosulfonated Cyanine Reference (7)

Example 5.1: Synthesis of a Non-Radioactive [¹⁹F]-Fluoro-MonosulfonatedCyanine Reference (7)

Cyanine amino-amide 6 (19.25 mg, 30.1 μmol) is dissolved in NMP (1 mL)and DIEA (180 μL of a 2.0 M solution in NMP, 360 μmol, 12 equiv.). 500μL of a 90 mM solution of NHS ester 1 in NMP is added and the resultingreaction mixture is stirred at room temperature overnight. The reactionis checked for completion by RP-HPLC (system B). Thereafter, thereaction mixture is diluted with aq. Tetraethylammonium bromide TEAB andpurified by semi-preparative RP-HPLC (system E, 1 injection,Rt=42.0-46.0 min). The product-containing fractions are lyophilised anddesalted by semi-preparative RP-HPLC (system F) to give the TFA salt offluoro-monosulfonated cyanine 7 (12 mg, 13 μmol, yield 43%) thenlyophilised.

HPLC (system B): Rt=26.0 min, purity>99%;

HPLC (system H): Rt=23.7 min.

Example 5.2: Synthesis of a Radioactive [¹⁸]-Monosulfonated Cyanine([¹⁸F]-7)

Cyanine amino-amide 6 is dissolved in CH₃CN containing 1% DIEA. Then,1.0 mL of the CH₃CN solution of [¹⁸F]-fluorosulfonated NHS ester [¹⁸F]-1(vide supra) is added. The vial is vigorously stirred at Roomtemperature for approximately one min. Thereafter, the reaction isstopped and directly analysed by RP-HPLC (system I with radioactivitydetection). HPLC (system I): tR=23.6 min. The retention time differencebetween the UV and radio traces (ca. 1 min) is caused by the serialarrangement of the detectors. This PET/fluorescent tracer is purified bySPE using an Oasis® HLB cartridge.

Example 6: Fluoro-Monosulfonated Dodecapeptide Reference (8)

Example 6.1: Synthesis of a Non-Radioactive [¹⁹F]-Fluoro-MonosulfonatedDodecapeptide ([¹⁹F]-8)

A dodecapeptide of sequence AcKGRANLRILARY is dissolved in H2O—CH3CN(1:1, v/v, 500 μL) and 2.6 μL of a 2.0 M solution of DIEA in NMP (5.2μmol, 4 equiv.) is added. 15 μL of a 90 mM solution of NHS ester 1 inNMP is added and the resulting reaction mixture is stirred at rtovernight. The reaction is checked for completion by RP-HPLC (system G).Thereafter, the reaction mixture is diluted with aq. TFA 0.1% andpurified by semi-preparative RP-HPLC (system H, 1 injection). Theproduct-containing fractions are lyophilised to give the TFA salt offluoro-monosulfonated dodecapeptide ([¹⁹F]-8).

HPLC (system A): Rt=21.3 mM, purity 96% (two diastereomers); HPLC(system I): Rt=15.2 min; λmax (recorded during the HPLC analysis)/nm261.

Example 6.2: Synthesis of a Radioactive [¹⁸F]-MonosulfonatedDodecapeptide ([¹⁸F]-8)

Dodecapeptide of sequence AcKGRANLRILARY is dissolved in watercontaining 1% DIEA. Then, 1.0 mL of the CH₃CN solution[¹⁸F]-fluorosulfonated NHS ester [¹⁸F]-1 (vide supra) is added. The vialis vigorously stirred at room temperature for less than one minute.Thereafter, the reaction is stopped and directly analysed by RP-HPLC(system I with radioactivity detection). HPLC (system I): tR=15.1 min.The peptide-based PET ([¹⁸F]-8) tracer is purified by SPE using anOasis® HLB cartridge.

The invention claimed is:
 1. A compound of formula (I) or apharmaceutically acceptable salt thereof:

in which the R⁰ bi-functional group is a spacer selected from the groupconsisting of the following radicals:

the R¹ monovalent group is selected from the group consisting of asuccinimidyl ester, a benzotriazole ester, a paranitrophenyl ester, aprotecting labile group and hydrogen; the R² monovalent group is ahydrogen, a metallic cation, an alkyl, a cyclo-alkyl, an aryl, anarylalkyl, an alkylaryl, an acyl, an alkenyl, an alkynyl radical or acombination of these radicals; the R^(2′) monovalent group is hydrogenor an alkyl, a cyclo-alkyl, an aryl, an arylalkyl, an alkylaryl, anacyl, an alkenyl, an alkynyl radical or a combination of these radicals;the R³ bi-functional group is moiety (CR⁴R⁵)_(n)—, wherein R⁴, R⁵ areeach individually selected from the group consisting of hydrogen, analkyl, a cycloalkyl, an aryl, an arylalkyl, an alkylaryl, an acyl, analkenyl, an alkynyl and a mixture thereof, wherein n is an integerbetween 1 and 3; and R^(*) is a (radio) nuclide.
 2. The compoundaccording to claim 1, wherein said compound to is represented by formula(II):


3. The compound according to claim 1, wherein said compound to isrepresented by formula (III):

wherein R⁴ and R⁵ are hydrogen.
 4. Drug or diagnosis product comprisingat least one compound according to claim 1, and a pharmaceuticallyacceptable carrier.